The present invention relates to: (1) DNA sequences encoding membrane-translocating peptide sequences: (2) sequences of membrane-translocating peptides; (3) fusion proteins endowing membrane-translocating potential upon biologically active polypeptides and proteins; and (4) expression vectors for production of said fusion proteins.
More specifically, the disclosed invention relates to a novel membrane-translocating sequence for directing import of biologically active protein molecules into a cell, and a method of using an expression vector in a host cell to produce a fusion protein comprising a membrane-translocating sequence and a biologically active polypeptide, protein domain, or protein.
Signal peptide sequences mediate protein secretion and are composed of a positively charged amino terminus, a central hydrophobic core and a carboxyl-terminal cleavage site recognized by a signal peptidase. These sequences usually comprise 15 to 30 residues. Signal sequences used for targeting proteins to specific locations have been found in both prokaryotic and eukaryotic cells. In bacteria, phage fd signal sequences for the major and minor coat proteins direct those proteins to the inner membrane. The xcex2-lactamase protein of pBR322 is directed to the periplasmic space by a different signal sequence, while outer membrane proteins such as OmpA are directed to their assigned destination by other signal sequences. Eukaryotic signal sequences directing translocation of the protein into the endoplasmic reticulum include that of human preproinsulin, bovine growth hormone, and the Drosophila glue protein. Near the N-terminus of such sequences are 2-3 polar residues, and within the signal sequence is a hydrophobic core consisting of hydrophobic amino acids. No other conservation of sequence has been observed (Lewin, 1994).
Peptide transport across the cell membrane has been demonstrated, for example, by a peptide representing the third helix of the Antennapedia homeodomain (Derossi et al., 1994). The transport peptide was not used to direct a cargo peptide through the cell membrane, however.
Biological membrane transport has been exploited for protein expression and export from transfected or transformed cells. Secretion of proteins, such as a globin protein, which would normally remain in the cytosol, has been achieved by adding a signal sequence to the N-terminus of the protein (Lewin 1994). Foreign genes have been inserted into recombinant DNA constructs for expression and secretion from bacterial cells, as described for example in U.S. Pat. No. 5,156,959, which discloses a method to export gene products into the growth medium of gram negative bacteria. U.S. Pat. No. 5,380,653 describes expression vectors and methods for intracellular protein production in Bacillus species. U.S. Pat. No. 5,712,114 describes a recombinant DNA construct for secretion of expressed proteins, particularly from Hansenula polymorpha cells, which utilizes the signal sequence of the human proprocollagen xcex1-1 protein.
Lin et al. have described a method of using a naturally-occurring signal peptide sequence to import a cargo peptide into the cell (Lin et al., 1995). One signal sequence that has been successfully used for this cell-permeable peptide import is the 16-residue h region of the signal sequence of Kaposi fibroblast growth factor. The cargo peptide transported by this technique has thus far been limited to no more than 25 amino acids, however.
Until now, DNA constructs, including both DNA vaccines and recombinant viral constructs, have provided the most effective method for furnishing a protein product to the cell for processing and expression of antigenic determinants on the cell surface. The Food and Drug Administration has expressed concern about approval of DNA vaccines, however, citing animal studies in which anti-DNA antibodies have been formed. Recombinant viral vectors have posed a unique set of problems in terms of delivery into cells, efficiency of expression, and potential immune system response to viral proteins. Other methods of DNA transfer into cells, such as transfection and microinjection, are often inefficient and time-consuming.
Genetic disorders resulting from the production of defective protein products have been treated, with limited success, by gene therapy. No other method has shown as much promise for introducing a protein into the interior of a cell. Gene therapy, however, has proven to be more difficult than originally envisioned. Appropriate vectors are difficult to identify, expression is transient, and immune responses to some vectors, particularly viral vectors, may preclude repeated use. Delivery of the isolated protein for import into the affected cells would provide a more efficient and effective solution to the problem.
What is needed is a method for importing entire protein molecules into a cell for studies of intracellular processes in living systems, for drug delivery, for vaccine development, and for disease therapy.
The present invention relates to a novel and non-naturally occurring membrane-translocating sequence (MTS) which has been shown to mediate the transport of a full-length protein into a cell. As used herein, a membrane-translocating sequence is an amino acid sequence capable of mediating the import of a polypeptide, protein domain, or full-length protein through the cell membrane.
The invention further relates to a method of using such a membrane-translocating sequence to genetically engineer proteins with cell membrane permeability. An expression vector is designed so that the DNA sequence encoding the membrane-translocating peptide will be positioned N-terminal or C-terminal to the sequence encoding the target protein, in correct reading frame for expression of both MTS and a biologically-active target protein as a fusion product. Peptides, polypeptides, protein domains, or full-length proteins are expressed as a fusion product with the membrane-translocating sequence.
Expression vectors may be chosen from among those readily available for prokaryotic or eukaryotic expression systems.
Genetically engineered proteins prepared by the method of the present invention can be used as protein-based vaccines, particularly where killed or attenuated whole organism vaccines are impractical.
Cell-permeable proteins prepared by the method of the present invention can also be used for the treatment of disease, particularly cancer. Cell-permeable proteins can be delivered to the interior of the cell, eliminating the need to transfect or transform the cell with a recombinant vector.
Cell permeable polypeptides of the present invention can be used in vitro to investigate protein function, or can be used to maintain cells in a desired state.
The membrane translocating sequence (MTS) of the present invention can be used to deliver peptides, polypeptides, protein domains, or proteins to the interior of a target cell either in vitro or in vivo. The MTS can be linked to the target protein through a peptide linkage formed by expression of the fusion protein from a recombinant DNA or RNA molecule, or can be linked to the target protein by means of a linker covalently linked to the MTS. A covalent linkage can be used to link an MTS of the present invention to a non-protein molecule, such as a polynucleotide, for import into the cell.